Variant of twin-arginine translocase and method for producing target protein using the same

Abstract

The present application provides a Tween-arginine translocation subunit TatC variant, and a microorganism containing said variant has excellent ability to secrete a target protein.

Description

Technology Field

[0001] The present application relates to a variant of a twin-arginine transport protein and a method for producing a target protein using the same. Background Technology

[0003] Microorganisms of the genus Corynebacterium have long been used in industrial fields for the production of amino acids and nucleic acids, and their safety has been recognized. Although these microorganisms possess a transport system to secrete proteins expressed within the cell to the outside, they are known to inherently possess a very limited variety of proteins secreted extracellularly, including proteases. Therefore, utilizing Corynebacterium microorganisms to establish a system for the extracellular secretion of target proteins facilitates the recovery and purification of the target proteins, resulting in high industrial utility.

[0004] Among the prior art studies that sought to utilize microorganisms of the genus Corynebacterium for the secretion and production of target proteins, there were also studies aimed at improving the secretion of target proteins [Korean Patent Publication 10-2022-0049827], but they were limited to enhancing the expression of some of the protein transport system genes inherently possessed by microorganisms of the genus Corynebacterium, and for this reason, there is a clear limitation in the effect of improving the secretion and production capacity of target proteins. Prior art literature

[0006] Republic of Korea Published Patent Application KR 10-2022-0049827 A The problem to be solved

[0007] The object of the present application is to include an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0008] (1) The amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids; or

[0009] (2) Provides a polypeptide in which the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids.

[0010] Another objective of the present application is to provide a polynucleotide encoding the polypeptide.

[0011] Another objective of the present application is to provide a recombinant vector comprising the polynucleotide.

[0012] Another object of the present application is the polypeptide and the target protein; or

[0013] The present invention provides a microorganism that produces a target protein, comprising a polynucleotide encoding the above polypeptide and a gene encoding the target protein.

[0014] Another objective of the present application is to provide a composition for producing a target protein comprising the microorganism.

[0015] Another objective of the present application is to provide a method for producing a target protein, comprising the step of culturing the microorganism in a culture medium. means of solving the problem

[0017] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not to be considered limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific aspects of this application described in this application using only ordinary experiments. Moreover, such equivalents are intended to be included in this application.

[0019] The present application will be described in more detail below.

[0021] polypeptide

[0022] The present application comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence of SEQ ID NO. 1; or the amino acid sequence of SEQ ID NO. 1, and

[0023] A polypeptide is provided in which the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1; or amino acid residues corresponding to the 106th, 107th and 108th residues are substituted with amino acids different from the original amino acids.

[0024] The above polypeptide may be a polypeptide having Tween-arginine transpotential enzyme activity.

[0025] The above polypeptide may be a Tween-arginine translocator subunit TatC protein.

[0026] In one example, the above-mentioned tween-arginine translocator subunit TatC protein may be of Corynebacterium glutamicum (WP_011014400.1) and may be represented by the amino acid sequence of SEQ ID NO. 1.

[0028] In one example, the polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0029] (1) The amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids; or

[0030] (2) The amino acid corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be a polypeptide in which the amino acid is substituted with another amino acid.

[0031] In one example, the polypeptide comprises the amino acid sequence of SEQ ID NO. 1, and

[0032] (1) The 106th and 107th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids; or

[0033] (2) The amino acid sequence of SEQ ID NO. 1 may be a polypeptide in which the 106th, 107th, and 108th amino acids from the N-terminus are substituted with other amino acids.

[0034] The amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1, or the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1, may be threonine.

[0035] Accordingly, the amino acid corresponding to the 106th residue or the 106th amino acid may be substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the amino acid corresponding to the 106th residue or the 106th amino acid may be substituted with alanine.

[0036] The amino acid corresponding to the 107th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1, or the 107th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1, may be proline.

[0037] Accordingly, the amino acid corresponding to the 107th residue or the 106th amino acid may be substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the amino acid corresponding to the 107th residue or the 106th amino acid may be substituted with serine.

[0038] The amino acid corresponding to the 108th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1, or the 108th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1, may be glycine.

[0039] Accordingly, the amino acid corresponding to the 108th residue or the 106th amino acid may be substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the amino acid corresponding to the 108th residue or the 106th amino acid may be substituted with alanine.

[0040] In one example, the above polypeptide is,

[0041] A sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0042] (1) In the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 106th residue from the N-terminus is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the amino acid corresponding to the 107th residue is substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or

[0043] (2) In the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 106th residue from the N-terminus is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, the amino acid corresponding to the 107th residue is substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the amino acid corresponding to the 108th residue is alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, It may be a polypeptide substituted with asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0044] In one example, the above polypeptide is,

[0045] A sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0046] (1) In the amino acid sequence of SEQ ID NO. 1, the amino acid corresponding to the 106th residue from the N-terminus is substituted with alanine and the amino acid corresponding to the 107th residue is substituted with serine; or

[0047] (2) The amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be replaced with alanine, the amino acid corresponding to the 107th residue may be replaced with serine, and the amino acid corresponding to the 108th residue may be replaced with alanine.

[0048] In one example, the polypeptide is,

[0049] (1) The 106th amino acid in the amino acid sequence of SEQ NO. 1 is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the 107th amino acid is substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or

[0050] (2) In the amino acid sequence of SEQ ID NO. 1, the 106th amino acid from the N-terminus is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, the 107th amino acid is substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the 108th amino acid is alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, It may be a polypeptide substituted with glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0051] In one example, the above polypeptide is,

[0052] (1) In the amino acid sequence of SEQ ID NO. 1, the 106th amino acid from the N-terminus is substituted with alanine and the 107th amino acid is substituted with serine; or

[0053] (2) The amino acid sequence of SEQ ID NO. 1 may be a polypeptide in which the 106th amino acid from the N-terminus is substituted with alanine, the 107th amino acid is substituted with serine, and the 108th amino acid is substituted with alanine.

[0054] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO. 1, and

[0055] (1) A polypeptide in which the 106th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO. 1 is substituted with alanine and the 107th amino acid is substituted with serine; or

[0056] (2) The amino acid sequence of SEQ ID NO. 1 may be a polypeptide in which the 106th amino acid from the N-terminus is substituted with alanine, the 107th amino acid is substituted with serine, and the 108th amino acid is substituted with alanine.

[0057] In one embodiment, the polypeptide may include the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO. 24, or be composed of the amino acid sequence.

[0058] It is obvious that if the polypeptide described above contains a variant in which amino acid residues corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1; or amino acid residues corresponding to the 106th, 107th, and 108th residues are substituted with amino acids different from the original amino acids, then other amino acid residues excluding said amino acid residues may be deleted, modified, substituted, or added, provided that they exhibit Tween-arginine translocation activity. For example, this may be the case where there are sequence additions or deletions that do not alter the activity of the polypeptide, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence of said polypeptide.

[0059] The aforementioned “conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0060] In one example, the polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0061] (1) The amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids; or

[0062] (2) In the amino acid sequence of SEQ ID NO. 1, the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus are substituted with other amino acids, and

[0063] It may be a polypeptide having Tween-arginine translocator activity.

[0064] In one example, the polypeptide having the above-mentioned twin-arginine translocator activity may be a twin-arginine translocator subunit TatC protein.

[0065] In one example, the polypeptide (e.g., a polypeptide having Tween-arginine translocator activity) or the Tween-arginine translocator subunit TatC protein may be of a microorganism of the genus Corynebacterium. The microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium epiphysiens ( Corynebacterium efficiens ), Corynebacterium calunae ( Corynebacterium callunae ), Corynebacterium stationaryis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), and Corynebacterium flavescens ( Corynebacterium flavescens It may be one or more microorganisms selected from a group consisting of ), but is not limited thereto.

[0066] In this application, the term "variant polypeptide" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence different from the amino acid sequence of the variant polypeptide before modification, while retaining functions or properties. Such variant polypeptides can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant polypeptide may be increased, unchanged, or decreased compared to the polypeptide before modification. Additionally, some variant polypeptides may include variant polypeptides in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variant polypeptides may include variant polypeptides in which a portion has been removed from the N- and / or C-terminus of a mature protein. The above term "variant polypeptide" may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English expressions, modification, modified polypeptide, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited to any term used in the sense of being mutated. Additionally, the variant polypeptide may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translational or post-translational protein translocation may be conjugated to the N-terminus of the variant polypeptide.In addition, the above variant polypeptide can be conjugated with other sequences or linkers so that it can be identified, purified, or synthesized.

[0067] For the purposes of the present application, the variant polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the amino acid sequence of SEQ ID NO. 1, and

[0068] (1) The amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with other amino acids; or

[0069] (2) The amino acid corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be a polypeptide in which the amino acid is substituted with another amino acid.

[0070] The above variant polypeptide may have increased activity that increases twin-arginine translocation activity and / or the ability of microorganisms to secrete and produce target proteins compared to the pre-mutation polypeptide (e.g., polypeptide containing the amino acid sequence of SEQ ID NO. 1).

[0072] polynucleotide

[0073] Another aspect provides a polynucleotide encoding the polypeptide.

[0074] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, and may mean a DNA or RNA strand of a certain length or longer.

[0075] The above polynucleotide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of sequence homology with the nucleic acid sequence of SEQ ID NO. 2, and

[0076] In the amino acid sequence of SEQ ID NO. 1, the codons encoding amino acid residues corresponding to the 106th and 107th residues from the N-terminus are substituted with codons encoding other amino acids, or

[0077] The polynucleotide may be one in which the codons coding for amino acid residues corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of SEQ ID NO. 1 are substituted with codons coding for other amino acids.

[0078] It was previously explained that the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1, or the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1, is threonine, the amino acid corresponding to the 107th residue, or the 107th amino acid is proline, and the amino acid corresponding to the 108th residue, or the 108th amino acid is glycine.

[0079] Accordingly, the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1 or the codon coding for the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1 may be ACU, ACC, ACA, or ACG, the amino acid corresponding to the 107th residue or the codon coding for the 107th amino acid may be CCU, CCC, CCA, or CCG, and the amino acid corresponding to the 108th residue or the codon coding for the 108th amino acid may be GGU, GGC, GGA, or GGG.

[0080] It is clearly known in the art what polynucleotide sequence the codon encoding each amino acid contains. The said codon may be modified in various ways without altering the amino acid sequence of the polypeptide, taking into account the degeneracy of the codon or the codons preferred by the organism intended to express the polypeptide.

[0081] In one embodiment, the polynucleotide may include the nucleic acid sequence of SEQ ID NO. 19 or SEQ ID NO. 25, or be composed of the nucleic acid sequence.

[0083] The polynucleotide of the present application may, without limitation, include probes that can be prepared from known gene sequences, for example, sequences that can be hybridized under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application. The “stringent condition” means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, under conditions in which polynucleotides with high homology or identity are hybridized with each other, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% or more of homology or identity, and polynucleotides with lower homology or identity are not hybridized with each other, or conventional Southern hybridization (southern Conditions for washing once, specifically two to three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions for hybridization) can be listed.

[0084] Hybridization requires that two nucleotides have complementary sequences, but hybridized polynucleotides may contain some mismatch between bases depending on the degree of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of this application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

[0085] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55 ℃ and using the conditions described above. Additionally, the Tm value may be 60 ℃, 63 ℃, or 65 ℃, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0086] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., i.e.).

[0087] In this specification, the phrase “a polynucleotide (which may be interchangeably used with “gene”) or a polypeptide (which may be interchangeably used with “protein”) “comprising a specific nucleic acid sequence or amino acid sequence or composed of or expressed by a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and may be interpreted as comprising a “substantially equivalent sequence” in which meaningless variations (deletion, substitution, modification, and / or addition) are applied to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or not excluding the meaningless variations).

[0088] In this specification, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0089] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0090] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0091] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0092] In this specification, the term “corresponding to” refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this application, “corresponding region” generally refers to a similar or corresponding position in a related protein or a reference protein.

[0093] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue of the said amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue of sequence number 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a "reference sequence").

[0094] For such alignment, examples such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) may be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art may be appropriately used.

[0096] recombination vector

[0097] Another aspect provides a recombinant vector containing the above polynucleotide.

[0098] In this specification, the term "vector" refers to a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, it may comprise, but is not limited to, a nucleic acid sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the target polypeptide in a suitable host cell. The control sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector may be maintained independently of the host cell's genome or inserted into the host cell's genome. For example, the target polynucleotide may be inserted into a chromosome via an insertion vector. The insertion of the above polynucleotide into a chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto.

[0099] The vectors available in this specification are not particularly limited as long as they are capable of replicating within a host cell and may be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc., in their natural or recombinant state. For example, as the vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, examples include pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc., but are not limited thereto.

[0100] The above vector may additionally include a selection marker to determine whether it is introduced into transformed cells or inserted into the genome of transformed cells. The selection marker is intended to confirm whether the cells transformed by the vector or the polynucleotide has been inserted, and may be selected from genes that confer selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0101] Expressing the above polypeptide in a microorganism can be performed by introducing a polynucleotide encoding the above polypeptide, or a vector containing the above polynucleotide, into a host cell and culturing a recombinant cell (e.g., a microorganism) containing the same.

[0102] The introduction of a polynucleotide encoding the above polypeptide or a vector containing said polynucleotide into a microorganism may be carried out by a person skilled in the art by appropriately selecting a known transformation method. In this specification, the term "transformation" means introducing a target polynucleotide or a vector containing said polynucleotide into a host cell (microorganism) to change the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into or located outside the chromosome of the host cell. The said polynucleotide may be introduced in an appropriate form depending on the purpose of introduction. For example, said polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. The expression cassette may include expression regulatory elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, which are typically operably linked to said polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" above may mean that the polynucleotide is functionally linked to an expression regulatory element (e.g., a promoter) so as to enable transcriptional regulation (e.g., transcription initiation) of the polynucleotide. Operable linkage can be performed using gene recombination techniques known in the art.

[0103] The method of transforming the above-mentioned polynucleotide into a host cell can be carried out by any method of introducing nucleic acid into a cell (microorganism), and depending on the host cell, transformation techniques known in the art can be appropriately selected. Examples of the above-mentioned known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG)-mediated uptake, DEAE-dextran method, cationic liposome method, lipofection, and lithium acetate-DMSO method.

[0104] The above recombinant vector may include the polynucleotide and a gene encoding the target protein.

[0105] The above-mentioned target protein may refer to any type of protein having biologically useful activity that is intended to be produced using the protein expression, secretion, and production system of microorganisms, and may refer to any protein that can be expressed in a host cell by inserting a polynucleotide encoding the said protein into a recombinant vector as a protein that a person skilled in the art intends to produce in large quantities. For example, the above-mentioned target protein may be collagen, collagen-derived polypeptide, hormone, hormone analog, cytokine, antigen, antigen-binding fragment, antibody, cell receptor, enzyme, transport protein, structural protein, serum, cell protein, antimicrobial peptide, antioxidant peptide, etc.

[0106] In one example, the target protein is collagen (collagen derived from, e.g., human, pig, cow, chicken, fish, etc.), collagen-derived polypeptide (a polypeptide containing a collagen-derived amino acid sequence), etanercept, epoetin alpha, infliximab, interferon alpha, insulin lispro, filgrastim, imiglucerase, glatiramer acetate, rituximab, pegfilgrastim, insulin grastim, adalimumab, trastuzumab, bevacizumab, ranibizumab, insulin, growth hormone, tumor necrosis factor (tumor necrosis factor-alpha), interleukin-7, insulin-like growth factor 2, interferon gamma, interferon alpha, interleukin-2, osteogenic protein, recombinant plasminogen-activator, bone morphogenetic protein 2, antifungal peptide, tissue plasminogen activator, immunoglobulin G, erythropoietin,granulocyte-macrophage stimulating factor, granulocyte-colony stimulating factor, muromomab, abciximab, daclizumab, basiliximab, palivizumab, ibritumomab, omalizumab, efalizumab, tositumomab, cetuximab, natalizumab, alkaline phosphatase (PhoA), levan fructotransferase (LFT), cellulose binding domain, cholera doxin B It may be one or more selected from the group consisting of (cholera toxin B), organophosphohydrolase, calcitonin, blood coagulation factors, hirudin, and monoclonal antibody 5T4, but is not limited thereto. In one example, the target protein may additionally include a tag for protein purification, expression, lysis, or detection enhancement of the target protein. Various tags that can be used for such purposes are known in the art, and may be, for example, GST tags, FLAG tags, polyarginine tags, polyhistidine tags, for example, 6His-tags, MBP tags, S-tags, influenza virus HA tags, thioredoxin tags, or Staphylococcus protein A tags.

[0107] In one example, the target protein is a bovine-derived collagen protein (bovine COL6A1, NP_001137337.1; SEQ ID NO. 5) or a polypeptide derived therefrom (SEQ ID NO. 6 or SEQ ID NO. 32) or Saccharomyces cerevisiae ( Saccharomyces cerevisiae It may be YGK peptide (Sequence No. 34), an antioxidant functional protein derived from ), but is not limited thereto.

[0108] In one example, the target protein may be a protein and / or peptide having a desired activity in the body (e.g., activity for the prevention, alleviation, and / or treatment of a specific disease or symptom, or activity for replacing a substance required by the body), and may be one or more selected from the group consisting of, for example, proteins or peptides of enzymatic activity (e.g., protease, kinase, phosphatase, etc.), receptor proteins or peptides, transporter proteins or peptides, bactericidal and / or endotoxin-binding polypeptides, structural proteins or peptides, immunopeptides, toxins, antibiotics, hormones, growth factors, vaccines, etc. In one example, the target polypeptide may be one or more selected from the group consisting of hormones, cytokines, tissue plasminogen activators, immunoglobulins (e.g., antibodies or their antigen-binding fragments or variants). The above immunoglobulin may be of any isotype (e.g., IgA, IgD, IgG, IgM, or IgE) and may be, for example, an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4). The above antigen-binding fragment is a fragment possessing the antigen-binding ability of the original antibody and may be any fragment of an antibody containing about 20 or more amino acids, for example, about 100 or more amino acids. The above antigen-binding fragment may be one or more selected from the group consisting of the antigen-binding site of the antibody, e.g., CDS, Fab fragment, F(ab)2 fragment, Fv, scFv, multibody containing multiple binding domains (e.g., diabody, triabody, tetrabody, etc.), single-domain antibody, affibody, etc. The above antibody variant is a derivative of an antibody or antibody fragment having the same binding function as the antibody but having an amino acid sequence modified from the original antibody.The antibody and / or antigen-binding fragment may be, for example, a mouse antibody, a human antibody, a chimeric antibody, a humanized antibody, or a human antibody. The antibody and / or antigen-binding fragment may be isolated from living organisms or may be of non-naturally occurring origin. The antibody and / or antigen-binding fragment may be produced synthetically or recombinantly. The antibody may be a monoclonal antibody. In another embodiment, the target polypeptide may be one or more selected from the group consisting of insulin, human growth hormone (hGH), insulin-like growth factor, EGF, VERF and various growth factors, various receptors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines (e.g., interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18), interferon (IFN)-alpha, -beta, -gamma, -omega or -tau, tumor necrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1, etc.

[0109] The recombinant vector may additionally include a gene encoding a secretion signal peptide. For the expression and secretion of a target protein through a microbial transport system, the target protein may be linked to a secretion signal peptide at its N' end. The recombinant vector may include a gene encoding a target protein and a gene encoding a secretion signal peptide in an appropriate order so as to express a form in which a secretion signal peptide is linked to the N' end of the target protein.

[0110] The above secretion signal peptides may be used without limitation as long as they are secretion signal peptides capable of inducing the secretion of a target protein in microorganisms, and in one example, Cg0955 secretion signal peptide, CgR0079 secretion signal peptide, CgR0120 secretion signal peptide, CgR0124 secretion signal peptide, CgR0900 secretion signal peptide, CgR0949 secretion signal peptide, CgR1023 secretion signal peptide, CgR1448 secretion signal peptide, CgR2137 secretion signal peptide, CgR2627 secretion signal peptide, CgR2926 secretion signal peptide, E. coli ( E. coli ) derived TorA secretory signaling peptide and Atrobacter globiformis ( Arthrobacter globiformis It may be one or more selected from the group consisting of IMD secretion signal peptides derived from ), but is not limited thereto. That is, any secretion signal peptide capable of inducing the secretion of a target protein in a microorganism may be used without limitation.

[0111] The recombinant vector may include a promoter operably linked to the gene encoding the target protein and / or the gene encoding the secretion signal peptide for the expression of the gene encoding the target protein and / or the gene encoding the secretion signal peptide.

[0112] In this specification, "promoter" may mean an untranscribed nucleotide sequence upstream or downstream of a coding region that includes a binding site for polymerase and has transcription initiation activity for a promoter target gene (e.g., a gene encoding a target protein or a Tween-arginine transposase, etc.), such as a DNA region to which polymerase binds to initiate transcription of the gene.

[0113] The above promoter may use any promoter sequence commonly used for gene expression, and examples of known promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent 10584338 B2), O2 promoter (US Patent 10273491 B2), tkt promoter, yccA promoter, etc.

[0114] In one example, the promoter may be used without limitation as long as it can regulate transcription initiation in a cell, e.g., a viral cell, a bacterial cell, a eukaryotic cell, an insect cell, a plant cell, or an animal cell. For example, the promoter may be one or more selected from the group consisting of, but not limited to, promoters of prokaryotic or mammalian viruses such as the CMV promoter (cytomegalovirus promoter), SV40 promoter, adenovirus promoter (major late promoter), pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, and HSV tk promoter, and animal cell promoters such as the metallothionin promoter and the beta-actin promoter.

[0115] The above recombinant vector may include one or more selected from the group consisting of a polynucleotide encoding a polypeptide provided in the present application, a gene encoding a target protein, and a gene encoding a secretion signal peptide.

[0116] The recombinant vector may include the polynucleotide, a gene encoding a target protein, and a gene encoding a secretion signal peptide.

[0117] The above polynucleotide, the gene encoding the target protein, and the gene encoding the secretion signal peptide can each be operably linked to the above promoter, and the promoter may be the same or different.

[0119] The polypeptide provided in this application may have enhanced Tween-arginine translocation activity. The polypeptide may have enhanced expression, secretion, and / or production capabilities of a target protein.

[0120] In this specification, the term “enhancement” of polypeptide activity means that the activity of a polypeptide within a host cell (microorganism) is increased compared to its intrinsic activity. The term “enhancement” may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The term “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic mutations due to natural or artificial factors. This may be used interchangeably with “activity prior to modification.” The statement that the activity of a polypeptide is “enhanced,” “upregulated,” “overexpressed,” or “increased” relative to its intrinsic activity means that it has been enhanced compared to the activity and / or concentration (expression amount) of a specific polypeptide originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0121] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be determined from the degree of activity, expression amount, or increase in the secretion and / or production of the target protein to which the secretory signal peptide recognized by the polypeptide is linked.

[0122] The enhancement of the activity of the above polypeptide may be achieved by applying various methods well known in the art, and is not limited to, as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may utilize, but is not limited to, gene engineering and / or protein engineering known to a person skilled in the art, which are routine methods of molecular biology (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0123] Specifically, the reinforcement of the polypeptide of the present application is

[0124] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0125] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0126] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0127] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0128] 5) Modification of the polynucleotide sequence encoding the polypeptide to enhance polypeptide activity (e.g., modification of the polynucleotide sequence of the polypeptide gene to code for a polypeptide modified to enhance polypeptide activity);

[0129] 6) Introduction of an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding the same;

[0130] 7) Codon optimization of polynucleotides encoding polypeptides;

[0131] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites; or

[0132] 9) It may be a combination of two or more selected from 1) to 8) above, but is not specifically limited thereto.

[0133] More specifically,

[0134] The increase in the intracellular copy number of the polynucleotide encoding the above 1) polypeptide may be achieved by introducing into a host cell a vector to which the polynucleotide encoding the said polypeptide is operably linked, which can replicate and function independently of the host. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the said polypeptide into the chromosomes within the host cell. The introduction into the chromosomes may be performed by introducing into the host cell a vector capable of inserting said polynucleotide into the chromosomes within the host cell, but is not limited thereto. The said vector is as described above.

[0135] Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide 2) above with a sequence having potent activity may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or may involve a sequence mutation, or replacement with a sequence having stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a potent promoter, but is not limited thereto.

[0136] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent No. 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, yccA promoter, etc.

[0137] The above 3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may, for example, be a substitution with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0138] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may involve the occurrence of sequence variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or may involve replacement with an amino acid sequence or polynucleotide sequence modified to have stronger activity or an amino acid sequence or polynucleotide sequence modified to increase activity, but is not limited thereto. Specifically, the replacement may be performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used in this case may additionally include a selection marker to confirm whether chromosome insertion has occurred. The selection marker is as described above.

[0139] The introduction of an exogenous polynucleotide exhibiting the activity of the polypeptide described in 6) above may be the introduction into a host cell of an exogenous polynucleotide encoding a polypeptide that exhibits the same or similar activity as the polypeptide. The exogenous polynucleotide is not limited in its origin or sequence as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction may be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the polypeptide may be generated and its activity increased by the expression of the introduced polynucleotide within the host cell.

[0140] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be a codon optimization of the intrinsic polynucleotide such that transcription or translation within the host cell increases, or a codon optimization of the extrinsic polynucleotide such that optimized transcription or translation occurs within the host cell.

[0141] 8) The above method of analyzing the tertiary structure of the polypeptide to select and modify or chemically modify an exposed site may involve, for example, determining a template protein candidate based on the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, confirming the structure based on this, and selecting and modifying or modifying an exposed site to be modified or chemically modified.

[0142] Such enhancement of polypeptide activity may involve increasing the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild type or the microorganism prior to modification, or increasing the amount of the product produced from said polypeptide, but is not limited thereto.

[0144] microorganism

[0145] Another aspect provides a microorganism comprising one or more selected from the group consisting of the polypeptide, the polynucleotide, and a vector comprising the polynucleotide.

[0146] In this application, the term "microorganism (or strain)" may include both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. Such microorganisms may be microorganisms in which specific mechanisms are enhanced or weakened due to causes such as the insertion of external genes or the enhancement or weakening of the activity of endogenous genes, and may include microorganisms that have undergone genetic modification for the production of a target protein. In this application, the terms "microorganism," "strain," "host," and "host cell" may be used interchangeably.

[0147] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having tween-arginine translocator activity, said tween-arginine translocator activity enhanced, having the ability to express, secrete, and / or produce a target protein, or having an increased ability to express, secrete, and / or produce a target protein.

[0148] As explained above, in the microorganism, the target protein may be linked to a secretion signal peptide.

[0149] In this application, "non-mutated microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to wild-type strains or natural-type strains themselves, or strains prior to genetic mutations caused by natural or artificial factors. For example, the "non-mutated microorganism" may, in one example, refer to a strain in which the variant polypeptide of this application or a polynucleotide encoding said variant polypeptide has not been introduced, or prior to such introduction. The "non-mutated microorganism" may be used interchangeably with "pre-mutation strain," "pre-mutation microorganism," "non-mutated strain," "non-mutated strain," "non-mutated microorganism," or "reference microorganism."

[0150] The above microorganism may be a microorganism of the genus Corynebacterium (Corynebacterium sp.). The above microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium epiphysiens ( Corynebacterium efficiens ), Corynebacterium calunae ( Corynebacterium callunae ), Corynebacterium stationaryis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli), Corynebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), and Corynebacterium flavescens ( Corynebacterium flavescens It may be one or more microorganisms selected from a group consisting of ), but is not limited thereto.

[0151] In one example, the target strain for comparing whether the secretion and production capacity of the target protein increases may be a wild-type genus of Corynebacterium containing the wild-type twin-arginine translocation enzyme subunit TatC protein (e.g., the amino acid sequence of SEQ ID NO. 1), such as the strain Corynebacterium glutamicum ATCC13032.

[0152] In one example, the microorganism with improved secretion and production capacity of the target protein may have an increased secretion and production capacity of the target protein of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, or 300% or more compared to the parent strain before mutation or the non-mutated microorganism.

[0153] In one example, the microorganism with enhanced secretion and production capabilities of the target protein has approximately the same secretion and production capabilities of the target protein as the parent strain before mutation or the non-mutated microorganism.

[0154] The above term “about” refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term “about.”

[0156] Composition for producing purpose protein and method of production

[0157] Another aspect is to provide a composition for producing a target protein comprising one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured.

[0158] Another aspect provides a use for the above microorganism for producing a target protein.

[0159] The composition of the present application may further include any suitable excipients commonly used in compositions for producing a target protein, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0161] Another aspect provides a method for producing (or manufacturing) a target protein, comprising the step of culturing the microorganism in a culture medium.

[0162] The method for producing the target protein of the present application may include the step of culturing the microorganism in a culture medium.

[0163] In this application, "culture" means growing the microorganism, such as a strain of Corynebacterium glutamicum, under appropriately controlled environmental conditions. The culture process may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0164] In this application, "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganism, such as a strain of Corynebacterium glutamicum, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing ordinary microorganisms without special limitations, but the microorganism of this application may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0165] Specifically, culture media for the above-mentioned microorganisms, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0166] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses (e.g., blackstrap molasses), rice husk, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0167] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0168] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0169] In addition, during the cultivation of the microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during cultivation, foam generation may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0170] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0171] The target protein produced by the culture of the present application may be secreted into the culture medium or remain within the cell.

[0172] The method for producing the protein of the purpose of the present application may additionally include, for example, the step of preparing the microorganism, the step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), prior to the culturing step.

[0173] The method for producing the target protein of the present application may further include a step of recovering the target protein from a culture medium (a culture medium in which the culture is performed) or a microorganism (e.g., a strain of the genus Corynebacterium) according to the culture. The recovery step may be additionally included after the culture step.

[0174] The above recovery may involve collecting the target protein using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the target protein may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0175] In addition, the method for producing the target protein of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for producing the target protein of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto. Effects of the invention

[0177] The present application provides a Tween-arginine translocation subunit TatC variant, and a microorganism containing said variant has excellent ability to secrete a target protein. Brief explanation of the drawing

[0179] Figure 1 shows the production concentration of the target protein (bCOL6_1) in Corynebacterium glutamicum strains expressing wild-type TatC protein or TatC variants containing amino acid single substitution variants (Lane 1: wild-type TatC protein; Lane 2: TatC T106A variant; Lane 3: TatC P107S variant; Lane 4: TatC G108A variant). Figure 2 shows the production concentration of the target protein (bCOL6_1) in Corynebacterium glutamicum strains expressing wild-type TatC protein or TatC variants containing amino acid combination substitution variants (Lane 1: wild-type TatC protein; Lane 2: TatC T106A / P107S variant; Lane 3: TatC P107S / G108A variant; Lane 4: TatC T106A / G108A variant; Lane 5: TatC T106A / P107S / G108A variant). Figure 3 shows the production concentration of the target protein (bCOL6_1) in Corynebacterium strains expressing Corynebacterium glutamicum TatC protein, E. coli-derived wild-type TatC protein, E. coli-derived TatC P97S variant, Bacillus subtilis-derived wild-type TatC protein, and Bacillus subtilis-derived TatC S95A / P96S / G97A variants (Lane 1: Corynebacterium glutamicum TatC protein; Lane 2: E. coli-derived wild-type TatC protein; Lane 3: E. coli-derived TatC P97S variant; Lane 4: Bacillus subtilis-derived wild-type TatC protein; Lane 5: Bacillus subtilis-derived TatC S95A / P96S / G97A variant). Figure 4 shows the production concentration of the target protein (bCOL6_1) in Corynebacterium glutamicum strains in which the gene encoding the TatC T106A / P107S variant or the TatC T106A / P107S / G108A variant was introduced into the chromosome (Lane 1: wild type; Lane 2: TatC T106A / P107S variant; Lane 3: TatC T106A / P107S / G108A variant). Figure 5 shows the production concentration of the target protein (bCOL6_2) in Corynebacterium glutamicum strains in which the gene encoding the TatC T106A / P107S variant or the TatC T106A / P107S / G108A variant was introduced into the chromosome (Lane 1: wild type; Lane 2: TatC T106A / P107S variant; Lane 3: TatC T106A / P107S / G108A variant). Figure 6 shows the production concentration of the target protein (YGK peptide) in Corynebacterium glutamicum strains in which the gene encoding the TatC T106A / P107S variant or the TatC T106A / P107S / G108A variant was introduced into the chromosome (Lane 1: wild type; Lane 2: TatC T106A / P107S variant; Lane 3: TatC T106A / P107S / G108A variant). Specific details for implementing the invention

[0180] The present invention will be explained in more detail below through the following examples. However, these are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0182] Example 1. Production of a target protein using a Corynebacterium glutamicum strain expressing a TatC gene containing a single amino acid or combined variant

[0183] The twin-arginine translocase (Tat) system, a protein transport system inherent in the Corynebacterium glutamicum strain, plays a role in secreting target proteins containing secretion signal peptide sequences recognized by this system to the outside of the cell membrane. To increase the production of target proteins using the Corynebacterium glutamicum strain, strategies were explored to enhance the secretion capacity of the Tat system. Among the components of the Tat system, the TatC protein is known to perform the role of a proofreader by recognizing the folding state of the target protein to be secreted and selecting and secreting only the protein with the correct folding state.

[0184] In the present application, a TatC variant was produced in which the amino acids of the 106th, 107th, and / or 108th residues of the Corynebacterium glutamicum TatC protein were substituted with other amino acids, and the effect on the secretion ability of the target protein of a Corynebacterium glutamicum strain expressing it was confirmed.

[0186] Example 1-1. Construction of a co-expression vector of the TatC gene containing a single amino acid variant and a model target protein

[0187] To compare the secretion and production capabilities of target proteins of Corynebacterium glutamicum strains expressing the Corynebacterium glutamicum TatC variant, an expression vector was constructed by simultaneously introducing a partial sequence of bovine collagen type 6 (bovine COL6A1) and Corynebacterium glutamicum TatC as model target proteins. Specifically, bovine COL6A1 sequence information [Sequence No. 5] (Registration No. NP_001137337.1) was obtained from the National Institutes of Health GenBank (NIH GenBank), and a C-terminal HisX6 tag (HHHHHH; Sequence No. 75) for protein purification was added to some of the amino acid sequences to name it bCOL6_1 [Sequence No. 6]. Based on the bCOL6_1 amino acid sequence, gene synthesis with the nucleotide sequence [Sequence No. 7] was carried out through codon optimization. PCR was performed using primers [SEQ No. 36] and [SEQ No. 37] with the synthesized gene DNA as a template, and the bCOL6_1 gene fragment was obtained. The PCR reaction was performed using Pfu-X DNA Polymerase (Solgent, Cat. No. SPX16-R500) and followed the manufacturer's protocol.

[0188] Genomic DNA of the wild-type Corynebacterium glutamicum ATCC13032 strain was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template with primers [SEQ No. 38] and [SEQ No. 39] to obtain the TatC gene fragment. The amino acid sequence of the TatC protein of the wild-type Corynebacterium glutamicum ATCC13032 strain is the amino acid sequence of SEQ No. 1 (WP_011014400.1), and the gene sequence coding for the said amino acid sequence is the nucleic acid sequence of SEQ No. 2.

[0189] To obtain the CgR0949 secretion signal peptide sequence [Sequence No. 8] recognized by the Tat system, PCR was performed using primers [Sequence No. 40] and [Sequence No. 41] with ATCC13032 genomic DNA as a template, and the CgR0949 secretion signal peptide fragment was obtained. To obtain the promoter sequence, PCR was performed using primers [Sequence No. 42] and [Sequence No. 43] with cj7 promoter (Pcj7) [Sequence No. 10] (US Patent 7662943 B2) as a template, and the Pcj7 fragment was obtained. In addition, to obtain the pyk promoter (Ppyk) sequence [Sequence No. 11], PCR was performed using primers [Sequence No. 44] and [Sequence No. 45] with the above ATCC13032 genomic DNA as a template, and the Ppyk fragment was obtained.

[0190] A recombinant vector was obtained by cloning the above-mentioned Pcj7 fragment, CgR0949 secretion signal peptide fragment, bCOL6_1 gene fragment, Ppyk fragment, TatC gene fragment, and the pCES208 vector ("Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes." Journal of microbiology and biotechnology 18.4 (2008): 639-647.) obtained by cutting with XbaI restriction enzyme using an In-fusion HD Cloning Kit (Takara Bio Inc, Cat. No. 638933) and named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(WT)”.

[0191] In order to obtain a TatC gene fragment containing a single amino acid variant, PCR was performed using the ATCC13032 genomic DNA as a template with primers [SEQN 38] and [SEQN 46] and primers [SEQN 47] and [SEQN 39], and gene fragments for the T106A variant in which threonine, the 106th amino acid of the TatC protein, was substituted with alanine were obtained. PCR was performed using primers [SEQ No. 38] and [SEQ No. 48] and primers [SEQ No. 49] and [SEQ No. 39] from the same template to obtain gene fragments for the P107S variant in which proline, the 107th amino acid of the TatC protein, was substituted with serine, and PCR was performed using primers [SEQ No. 38] and [SEQ No. 50] and primers [SEQ No. 51] and [SEQ No. 39] to obtain gene fragments for the G108A variant in which glycine, the 108th amino acid of the TatC protein, was substituted with alanine. Recombinant vectors were obtained by cloning the individual amino acid variant gene fragments of the above TatC, the Pcj7 fragment, the CgR0949 secretion signal peptide fragment, the bCOL6_1 gene fragment, the Ppyk fragment, and the pCES208 vector cleaved with XbaI restriction enzyme using the In-fusion HD Cloning Kit, and were named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(P107S)”, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(G108A)”, respectively.

[0192] The amino acid sequences of the above TatC T106A variant, TatC P107S variant, and TatC G108A variant are the amino acid sequences of SEQ ID NO. 12, SEQ ID NO. 14, and SEQ ID NO. 16, respectively, and the gene sequences coding for the above amino acid sequences are the nucleic acid sequences of SEQ ID NO. 13, SEQ ID NO. 15, and SEQ ID NO. 17, respectively.

[0194] Example 1-2. Production of a target protein using a Corynebacterium glutamicum strain expressing the TatC gene containing a single amino acid mutation

[0195] The recombinant vectors prepared in Example 1-1 above were transformed into the wild-type ATCC13032 strain of Corynebacterium glutamicum by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-tatC(WT)”, “ATCC13032-bCOL6_1-tatC(T106A)”, “ATCC13032-bCOL6_1-tatC(P107S)”, and “ATCC13032-bCOL6_1-tatC(G108A)”, respectively.

[0196] A culture evaluation was conducted to confirm the target protein secretion and production capabilities of the four Corynebacterium glutamicum strains mentioned above. Specifically, after subculturing colonies of each strain in nutrient medium, each strain was inoculated into a 250 ml Corner-Baffle flask containing 25 ml of production medium and cultured at 30°C for 48 hours with shaking at 200 rpm. Each medium contained 50 mg / L of the antibiotic kanamycin to maintain the recombinant vector.

[0197] [Nutrient medium (pH 7.2)]

[0198] Glucose 10g, meat extract 5g, polypeptide 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g, kanamycin 50mg (based on 1 liter of distilled water)

[0199] [Production Medium (pH 7.0)]

[0200] Glucose 60g, Ammonium sulfate 30g, Yeast extract 10g, Potassium diphosphate 1g, Magnesium sulfate heptahydrate 0.4g, Iron heptahydrate 20mg, Manganese sulfate pentahydrate 1.8mg, Biotin 1.8mg, Thiamine-HCl 9mg, Calcium carbonate 50g, Kanamycin 50mg (based on 1 liter of distilled water)

[0202] After culture was completed under the above conditions, the production concentration of the bCOL6-1 target protein was confirmed through analysis of the culture supernatant. Specifically, the culture medium was centrifuged at 5000xg for 10 minutes to allow the cells to settle, and only the culture supernatant was separated and subjected to electrophoresis on a 4-20% Tris-Glycine SDS Precast Gel (Labis Koma, Cat. No. KG75355) using the SDS-PAGE method (Laemmli, 1970). The gel was stained using DIRECTBLUE™ gel staining solution (SCGBIOMAX, Cat. No. BDS-1000), images of the stained gel were acquired using a Calibrated Densitometer (BIO-RAD, GS-900), and the concentration of the target protein in the culture medium was analyzed. In addition, LC-MS analysis using the in-gel digestion method (Rosenfeld, 1992) was performed to confirm whether the amino acid sequence contained in the protein band of the corresponding size matched the target protein. The production concentrations of the bCOL6-1 target protein of the four strains above are shown in Figure 1 and Table 1 below.

[0204] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-bCOL6_1-tatC(WT) 279.8 - 2 experimental group ATCC13032-bCOL6_1-tatC(T106A) 165.7 -40.78% 3 ATCC13032-bCOL6_1-tatC(P107S) 202.2 -27.73% 4 ATCC13032-bCOL6_1-tatC(G108A) 228.9 -18.19%

[0205] As shown in Table 1 above, the strain expressing the TatC variant containing a single amino acid substitution variant did not show an increase in the concentration of the target protein (bCOL6_1) compared to the control strain expressing the wild-type TatC protein (ATCC13032-bCOL6_1-tatC(WT)).

[0207] Examples 1-3. Production of a target protein using a Corynebacterium glutamicum strain expressing a TatC gene containing an amino acid integration variant

[0208] Recombinant vectors were constructed to evaluate the effect of combining the amino acid single substitution variant of Example 1-1 on the secretion and production capacity of the target protein of the Corynebacterium glutamicum strain.

[0209] Specifically, using the genomic DNA of the above-mentioned Corynebacterium glutamicum ATCC13032 strain as a template, PCR was performed using primers [SEQN 38] and [SEQN 52], and primers [SEQN 53] and [SEQN 39] to obtain gene fragments for the T106A and P107S combined variant. From the same template, PCR was performed using primers [SEQN 38] and [SEQN 54], and primers [SEQN 55] and [SEQN 39] to obtain gene fragments for the P107S and G108A combined variant, and PCR was performed using primers [SEQN 38] and [SEQN 56], and primers [SEQN 57] and [SEQN 39] to obtain gene fragments for the T106A and G108A combined variant. Likewise, PCR was performed using primers [Sequence No. 38] and [Sequence No. 58] and primers [Sequence No. 59] and [Sequence No. 39] from the same template, and gene fragments for T106A, P107S, and G108A combined variants were obtained.

[0210] Recombinant vectors were obtained by cloning the amino acid-integrated variant gene fragments of the above TatC, the Pcj7 fragment obtained in Example 1-1, the CgR0949 secretion signal peptide fragment, the bCOL6_1 gene fragment, the Ppyk fragment, and the pCES208 vector cleaved with XbaI restriction enzyme using the In-fusion HD Cloning Kit, respectively, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(P107S / G108A)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / G108A)”, It was named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S / G108A)”.

[0211] The amino acid sequences of the above TatC T106A / P107S variant, TatC P107S / G108A variant, TatC T106A / G108A variant, and TatC T106A / P107S / G108A variant are the amino acid sequences of SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, and SEQ ID NO. 24, respectively, and the gene sequences coding for the above amino acid sequences are the nucleic acid sequences of SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, and SEQ ID NO. 25, respectively.

[0213] The recombinant vectors produced above were transformed into wild-type Corynebacterium glutamicum ATCC13032 strains by electroporation, and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-tatC(T106A / P107S)”, “ATCC13032-bCOL6_1-tatC(P107S / G108A)”, “ATCC13032-bCOL6_1-tatC(T106A / G108A)”, and “ATCC13032-bCOL6_1-tatC(T106A / P107S / G108A)”, respectively. In order to compare the secretion and production capabilities of the target protein of the above four types of Corynebacterium glutamicum strains and the ATCC13032-bCOL6_1-tatC(WT) strain obtained in Example 1-2, the strains were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture medium was analyzed and is shown in Figure 2 and Table 2 below.

[0214] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-bCOL6_1-tatC(WT) 278.6 - 2 experimental group ATCC13032-bCOL6_1-tatC(T106A / P107S / G108A) 771.3 176.85% 3 ATCC13032-bCOL6_1-tatC(T106A / P107S) 781.2 180.4% 4 ATCC13032-bCOL6_1-tatC(P107S / G108A) 201 -27.85% 5 ATCC13032-bCOL6_1-tatC(T106A / G108A) 267.2 -4.09%

[0215] As shown in Table 2 above, among the strains expressing TatC variants containing amino acid combination substitution variants, the ATCC13032-bCOL6_1-tatC(T106A / P107S) strain and the ATCC13032-bCOL6_1-tatC(T106A / P107S / G108A) strain showed a target protein concentration approximately 2.8 times higher compared to the control strain expressing wild-type TatC protein (ATCC13032-bCOL6_1-tatC(WT)).

[0217] Example 2. Production of a target protein using a Corynebacterium glutamicum strain expressing an alien microorganism-derived TatC gene containing an amino acid integration variant

[0218] The results of Example 1 above indicated that the T106A / P107S and T106A / P107S / G108A variants of Corynebacterium glutamicum TatC improved the secretion and production capacity of the target protein of the Corynebacterium glutamicum strain. Extending this, it was confirmed whether the same improvement in the secretion and production capacity of the target protein is exhibited when equivalent variants are applied to TatC genes derived from foreign microorganisms. Specifically, amino acid sequence information [SEQN 3; WP_000109943.1] coding for the TatC gene of Escherichia coli [SEQN 4] and amino acid sequence information [SEQN 26; coding for the TatC gene of Bacillus subtilis [SEQN 27] from the National Institutes of Health Gen Bank [WP_010886426.1] was obtained, and homology analysis was performed using the National Institutes of Health BLAST with the amino acid sequence information [SEQN 1; WP_011014400.1] coded by the TatC gene of Corynebacterium glutamicum [SEQN 2]. From the results, it was confirmed that proline, the 97th amino acid of the E. coli-derived TatC protein, and proline, the 96th amino acid of the Bacillus subtilis-derived TatC protein, correspond to proline, the 107th amino acid of the Corynebacterium glutamicum-derived TatC. Since the 96th and 98th amino acids of the E. coli-derived TatC protein are alanine, a TatC P97S variant was constructed by exclusively substituting the 97th amino acid, proline, with serine. Additionally, S95A / P96S / G97A variants were constructed by substituting the 95th amino acid, serine, with alanine, the 96th amino acid, proline, with serine, and the 97th amino acid, glycine, with alanine of the Bacillus subtilis-derived TatC protein. Specifically, genomic DNA from E. coli strain K-12 KCTC 2223 and Bacillus subtilis strain KCTC 3135 was extracted using a G-spin Total DNA Extraction Mini Kit according to the protocol provided in the kit.PCR was performed using primers [SEQ No. 60] and [SEQ No. 61] with the genomic DNA of the above-extracted Escherichia coli K-12 KCTC 2223 strain as a template to obtain wild-type Escherichia coli TatC gene fragments, and PCR was performed using primers [SEQ No. 60] and [SEQ No. 62] and primers [SEQ No. 63] and [SEQ No. 61] from the same template to obtain gene fragments for the Escherichia coli TatC P97S variant. Likewise, PCR was performed using primers [SEQN 64] and [SEQN 65] with the genomic DNA of the above-extracted Bacillus subtilis KCTC 3135 strain as a template to obtain wild-type Bacillus subtilis TatC gene fragments, and PCR was performed using primers [SEQN 64] and [SEQN 66] and primers [SEQN 67] and [SEQN 65] from the same template to obtain gene fragments for the Bacillus subtilis TatC S95A / P96S / G97A variant.

[0219] Recombinant vectors were obtained by cloning the wild-type gene fragments and P97S variant gene fragments of the above-mentioned Escherichia coli TatC, the wild-type gene fragments and S95A / P96S / G97A variant gene fragments of Bacillus subtilis TatC, the Pcj7 fragment, CgR0949 secretion signal peptide fragment, bCOL6_1 gene fragment, Ppyk fragment obtained in Example 1-1, and the pCES208 vector cleaved with XbaI restriction enzyme using the In-fusion HD Cloning Kit, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_EctatC(WT)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_EctatC(P97S)”, respectively. It was named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_BstatC(WT)” and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_BstatC(S95A / P96S / G97A)”.

[0220] The amino acid sequences of the above-mentioned E. coli-derived TatC P97S variant and the Bacillus subtilis-derived TatC S95A / P96S / G97A variant are the amino acid sequences of SEQ ID NO. 28 and SEQ ID NO. 30, respectively, and the gene sequences coding for the above-mentioned amino acid sequences are the nucleic acid sequences of SEQ ID NO. 29 and SEQ ID NO. 31, respectively.

[0222] The recombinant vectors produced above were transformed into the wild-type ATCC13032 strain of Corynebacterium glutamicum by electroporation, and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-EctatC(WT)”, “ATCC13032-bCOL6_1-EctatC(P97S)”, “ATCC13032-bCOL6_1-BstatC(WT)”, and “ATCC13032-bCOL6_1-BstatC(S95A / P96S / G97A)”, respectively. In order to compare the secretion and production capabilities of the target protein of the above four types of Corynebacterium glutamicum strains and the ATCC13032-bCOL6_1-tatC(WT) strain obtained in Examples 1-2, the strains were cultured in the same manner as in Examples 1-2, and the concentration of the target protein in the culture medium was analyzed and is shown in Figure 3 and Table 3 below.

[0223] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-bCOL6_1-tatC(WT) 266.3 - 2 experimental group ATCC13032-bCOL6_1-EctatC(WT) 116.1 -56.40% 3 ATCC13032-bCOL6_1-EctatC(P97S) 116.5 -56.25% 4 ATCC13032-bCOL6_1-BstatC(WT) 164.5 -38.23% 5 ATCC13032-bCOL6_1-BstatC(S95A / P96S / G97A) 179.3 -32.67%

[0224] As shown in Table 3 above, it was confirmed that the Corynebacterium glutamicum strain expressing Escherichia coli TatC (ATCC13032-bCOL6_1-EctatC(WT)) or the Corynebacterium glutamicum strain expressing Bacillus subtilis TatC (ATCC13032-bCOL6_1-BstatC(WT)) showed lower target protein concentrations compared to the control strain expressing Corynebacterium glutamicum TatC (ATCC13032-bCOL6_1-tatC(WT)). In addition, the Corynebacterium glutamicum strain into which the E. coli TatC P97S variant was introduced, with an amino acid substitution variant corresponding to the P107S variant of Corynebacterium glutamicum TatC introduced (ATCC13032-bCOL6_1-EctatC(P97S)), and the Corynebacterium glutamicum strain into which the Bacillus subtilis TatC S95A / P96S / G97A variant was introduced, with an amino acid substitution variant corresponding to the T106A / 9107S / G108A variant of Corynebacterium glutamicum TatC introduced (ATCC13032-bCOL6_1-BstatC(S95A / P96S / G97A)), respectively, are Corynebacterium glutamicum strains expressing wild-type E. coli TatC and strains expressing wild-type Bacillus subtilis TatC No significant increase in the target protein concentration was observed compared to the Corynebacterium glutamicum strain.

[0225] These results indicate that the effect of improving the secretion and production capacity of the target protein by the Corynebacterium glutamicum TatC variant confirmed in Examples 1-3 is not universally observed in TatC derived from foreign microorganisms.

[0227] Example 3. Production of an improved strain of Corynebacterium glutamicum in which a TatC gene containing amino acid integration variants is introduced into the chromosome, and production of various target proteins using the same

[0228] An improved strain was constructed by introducing the Corynebacterium glutamicum TatC T106A / P107S variant or the TatC T106A / P107S / G108A variant, which was confirmed to improve the secretion and production ability of the target protein of the Corynebacterium glutamicum strain from the results of Example 1 above, into the chromosome. Specifically, PCR was performed using primers [SEQ ID NO. 68] and [SEQ ID NO. 69] with the above-mentioned pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S) or pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S / G108A) vector DNA as a template, and gene fragments for the TatC T106A / P107S variant and the TatC T106A / P107S / G108A variant were obtained, respectively. Recombinant vectors were obtained by cloning the above-mentioned TatC integrated variant gene fragment and the pDZ vector (US Patent No. 9109242 B2) cut with XbaI restriction enzyme using the In-fusion HD Cloning Kit, and these were introduced onto the chromosome through homologous recombination by transforming the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation. Subsequently, improved strains in which the wild-type TatC gene on the chromosome was replaced with the integrated variant gene were identified by PCR using primers [SEQ No. 68] and [SEQ No. 69] on colonies that had undergone a second crossover process, and were named “ATCC13032::tatC(T106A / P107S)” and “ATCC13032::tatC(T106A / P107S / G108A)”, respectively.

[0229] To compare the secretion and production capabilities of the target protein in a Corynebacterium glutamicum strain into which the TatC variant was introduced, an expression vector for the bCOL6_1 target protein was constructed. Specifically, PCR was performed using primers [SEQN 42] and [SEQN 70] with the DNA of the pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(WT) vector as a template, and a gene fragment for the Pcj7_CgR0949_bCOL6_1 expression cassette was obtained. A recombinant vector was obtained by cloning the gene fragment and the pCES208 vector, which had been cleaved with XbaI restriction enzyme, using an In-fusion HD Cloning Kit, and was named “pCES208-Pcj7_CgR0949_bCOL6_1”. The wild-type strain of Corynebacterium glutamicum ATCC13032 and the improved strains ATCC13032::tatC(T106A / P107S) and ATCC13032::tatC(T106A / P107S / G108A) were transformed with the pCES208-Pcj7_CgR0949_bCOL6_1 recombinant vector by electroporation, and the strains into which the recombinant vector was introduced were named “ATCC13032-bCOL6_1”, “ATCC13032::tatC(T106A / P107S)-bCOL6_1”, and “ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1”, respectively. To compare the secretion and production capabilities of the target protein of the three Corynebacterium glutamicum strains mentioned above, they were cultured in the same manner as in Examples 1-2 above, and the concentration of the target protein in the culture medium was analyzed and is shown in Figure 4 and Table 4 below.

[0230] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-bCOL6_1 85.3 - 2 experimental group ATCC13032::tatC(T106A / P107S)-bCOL6_1 193.1 126.38% 3 ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1 188.4 120.87%

[0231] As shown in Table 4 above, it was confirmed that the Corynebacterium glutamicum strain with the Corynebacterium glutamicum TatC T106A / P107S variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S)-bCOL6_1) and the Corynebacterium glutamicum strain with the Corynebacterium glutamicum TatC T106A / P107S / G108A variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1) exhibited a target protein concentration more than twice as high compared to the control strain (ATCC13032-bCOL6_1).

[0232] From these results, it was confirmed that Corynebacterium glutamicum strains expressing the TatC T106A / P107S variant or the T106A / P107S / G108A variant introduced into the chromosome exhibited enhanced target protein secretion and production capabilities.

[0234] To verify whether the effect of enhancing target protein secretion and production capacity exhibited by Corynebacterium glutamicum strains into which the TatC variant was introduced into the chromosome is similarly observed for other target proteins, expression vectors for target proteins other than bCOL6_1 were constructed. Specifically, a C-terminal HisX6 tag (HHHHHH; SEQ ID NO. 75) for protein purification was added to some amino acid sequences in the bovine COL6A1 sequence information that differ from bCOL6_1, and the resulting vector was named bCOL6_2 [SEQ ID NO. 32]. Following codon optimization, gene synthesis was carried out with the nucleic acid sequence [SEQ ID NO. 33]. PCR was performed using the synthesized DNA as a template and primers [SEQ ID NO. 71] and [SEQ ID NO. 72] to obtain the bCOL6_2 gene fragment. Meanwhile, Saccharomyces cerevisiae ( Saccharomyces cerevisiaeTo construct an expression vector for the YGK peptide (containing a C-terminal HisX6 tag), an antioxidant functional protein derived from [Sequence No. 34] [Mirzaei et al., Journal of Functional Foods, 2015, Journal of Functional Foods Volume 19, Part A, December 2015, Pages 259-268], gene synthesis with the nucleic acid sequence of [Sequence No. 35] was carried out after codon optimization, and PCR was performed using primers of [Sequence No. 73] and [Sequence No. 74] with the synthesized DNA as a template to obtain the YGK gene fragment. Recombinant vectors were obtained by cloning each of the above gene fragments, the Pcj7 fragment obtained in Example 1-1, the CgR0949 secretion signal peptide fragment, and the pCES208 vector cleaved with XbaI restriction enzyme using the In-fusion HD Cloning Kit, and were named “pCES208-Pcj7_CgR0949_bCOL6_2” and “pCES208-Pcj7_CgR0949_YGK”, respectively.

[0235] The wild-type Corynebacterium glutamicum ATCC13032 strain and the improved strains ATCC13032::tatC(T106A / P107S) and ATCC13032::tatC(T106A / P107S / G108A) were transformed with the recombinant vectors by electroporation, and the strains into which the recombinant vectors were introduced were “ATCC13032-bCOL6_2”, “ATCC13032::tatC(T106A / P107S)-bCOL6_2”, “ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2”, “ATCC13032-YGK”, and “ATCC13032::tatC(T106A / P107S)-YGK”, respectively. It was named “ATCC13032::tatC(T106A / P107S / G108A)-YGK”.

[0236] First, in order to compare the secretion and production capabilities of the target protein of the three Corynebacterium glutamicum strains introduced with the bCOL6_2 expression vector, they were cultured in the same manner as in Examples 1-2 above, and the concentration of the target protein in the culture medium was analyzed and is shown in Figure 5 and Table 5 below.

[0237] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-bCOL6_2 110.6 - 2 experimental group ATCC13032::tatC(T106A / P107S)-bCOL6_2 176.5 59.59% 3 ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2 165.8 49.91%

[0238] As shown in Table 5 above, it was confirmed that the Corynebacterium glutamicum strain with the TatC T106A / P107S variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S)-bCOL6_2) and the Corynebacterium glutamicum strain with the TatC T106A / P107S / G108A variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2) exhibited a target protein concentration approximately 1.5 times higher for the bCOL6_2 target protein compared to the control strain (ATCC13032-bCOL6_2).

[0240] Next, in order to compare the secretion and production capabilities of the target protein of the three Corynebacterium glutamicum strains introduced with the YGK expression vector, they were cultured in the same manner as in Examples 1-2 above, and the concentration of the target protein in the culture medium was analyzed and is shown in Figure 6 and Table 6 below.

[0241] number division strain name Target protein concentration (mg / L) Change (%) compared to the control group 1 control group ATCC13032-YGK 49.4 - 2 experimental group ATCC13032::tatC(T106A / P107S)-YGK 134.0 171.26% 3 ATCC13032::tatC(T106A / P107S / G108A)-YGK 148.6 200.81%

[0242] As shown in Table 6 above, it was confirmed that the Corynebacterium glutamicum strain with the TatC T106A / P107S variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S)-bCOL6_2) and the Corynebacterium glutamicum strain with the TatC T106A / P107S / G108A variant introduced into the chromosome (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2) exhibited target protein concentrations 2.7 times and 3 times higher, respectively, for the YGK target protein compared to the control strain (ATCC13032-bCOL6_2).

[0243] From the above results, it was confirmed that Corynebacterium glutamicum strains in which the TatC T106A / P107S variant or the TatC T106A / P107S / G108A variant was introduced into the chromosome exhibited enhanced target protein secretion and production capabilities for various sequences and types of target proteins.

[0245] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A polypeptide comprising the amino acid sequence of SEQ ID NO. 1, (1) in which the amino acid corresponding to the 106th residue from the N-terminus of the amino acid sequence of SEQ ID NO. 1 is substituted with alanine and the amino acid corresponding to the 107th residue is substituted with serine; or (2) in which the amino acid corresponding to the 106th residue from the N-terminus of the amino acid sequence of SEQ ID NO. 1 is substituted with alanine, the amino acid corresponding to the 107th residue is substituted with serine and the amino acid corresponding to the 108th residue is substituted with alanine. Claim 2 delete Claim 3 delete Claim 4 In claim 1, the polypeptide is a polypeptide having twin-arginine translocase activity. Claim 5 In claim 1, the polypeptide is a polypeptide that is a twin-arginine transpotential enzyme subunit TatC protein. Claim 6 In claim 1, the polypeptide is a polypeptide derived from a microorganism of the genus Corynebacterium. Claim 7 In paragraph 6, the above-mentioned microorganism of the genus Corynebacterium is a polypeptide, which is Corynebacterium glutamicum. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 In claim 1, a polypeptide comprising the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO.

24. Claim 12 A polynucleotide encoding the polypeptide of any one of paragraphs 1, 4 through 7 and 11. Claim 13 A recombinant vector comprising the polynucleotide of claim 12. Claim 14 A recombinant vector comprising, in addition to, a gene encoding a target protein in claim 13. Claim 15 delete Claim 16 A polypeptide and a target protein of claim 1; or a microorganism producing a target protein comprising a polynucleotide encoding the polypeptide and a gene encoding the target protein. Claim 17 In paragraph 16, the microorganism is a microorganism with enhanced ability to secrete and produce the target protein. Claim 18 In paragraph 16, the microorganism is a microorganism of the genus Corynebacterium. Claim 19 In paragraph 18, the microorganism of the genus Corynebacterium is a microorganism that is Corynebacterium glutamicum. Claim 20 A composition for producing a target protein comprising one or more selected from the group consisting of a microorganism of any one of claims 16 to 19 and a culture medium in which said microorganism is cultured. Claim 21 A method for producing a target protein, comprising the step of culturing a microorganism of any one of claims 16 to 19 in a culture medium. Claim 22 A method for producing a target protein according to claim 21, further comprising, after the culturing step, a step of recovering the target protein from the cultured microorganism, the medium, or both.

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